# 2.2.8 研究 | 2021-多组学分析肺结核队列的记忆T细胞状态 ## 速览 * 题目：Multimodally profiling memory T cells from a tuberculosis cohort identifies cell state associations with demographics, environment and disease * 作者：哈佛医学院 Soumya Raychaudhuri 团队 > 首先感觉他们实验室主页做的很不错： ![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-061211.png) * 链接： * 发表在：Nature Immunology （2021-05-24） * 数据是公开的：[GSE158769](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE158769) ，采用CITE-seq ``` Platforms GPL11154 Illumina HiSeq 2000 (Homo sapiens) Samples GSM4810298 Memory_T_cell_CITE-seq GSE158769_exprs_norm.tsv.gz 3.0 Gb (ftp)(http) TSV GSE158769_exprs_raw.tsv.gz 747.0 Mb (ftp)(http) TSV GSE158769_meta_data.txt.gz 19.8 Mb (ftp)(http) TXT ``` Genotype data：[phs002025.v1.p1](https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs002025.v1.p1) * 分析代码： ![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-082342.png) * 还发开了一个网页： ## 摘要 * 分析了来自秘鲁的259个肺结核（tuberculosis , TB)病人的500,089个记忆T细胞，得到31种细胞状态 * 在排除年龄、性别、季节、遗传等因素影响后，发现了一种多能型type 17 helper T (TH17) 细胞状态，在结核杆菌发展成肺结核病的过程中丰度和功能都有所下降 ## 方法 **数据准备** * PBMC sample preparation * Flow cytometry of total PBMCs * CITE-seq of isolated memory T cells * Bulk RNA sequencing * Genotyping and genetic data processing **数据分析** * scRNA比对+定量 * scRNA数据拆分 * scRNA数据质控 * 降维 * 聚类+注释 * bulk RNA-seq 比对+定量 * 利用MASC(modeling of associations of single cells )评价不同细胞状态与疾病的关系 ## 先用CITE-seq测了>500,000个T细胞 * 在秘鲁首都利马进行了一次大型的流行病学调查（n= 18,544），从其中招募了264个人，采样了外周血单核细胞进行CITE-seq测定，同时还加入了基因表型数据和蛋白数据 * 图b是进行了6次QC，每一次过滤掉一些细胞，最后得到了500K * 大部分样本的细胞数量在2k左右 ![](https://media.springernature.com/full/springer-static/image/art%3A10.1038%2Fs41590-021-00933-1/MediaObjects/41590_2021_933_Fig1_HTML.png) ## 得到记忆T细胞的31种不同状态 利用canonical correlation analysis (CCA) 进行降维，选择top20 canonical variates (CVs)进行批次校正和聚类，根据基因和蛋白marker得到31个细胞状态 * 23/31 were CD4+; five were CD8+; one (C-24) was a mixture; Two clusters (C-30 and C-31) were CD4−CD8− * **C-20**： contained a subset of CD4−CD8− and CD8+ cells expressing innate-like T cell markers, including ZBTB16 and CD161 and CD26 surface proteins * one central memory cluster (**C-25**) and distinct *GZMK*+ (**C-28**) and *GZMB*+ (**C-29**) effector subsets, reflecting different cytotoxicities * high expression of HLA-DR and CD38 surface protein and proliferation-associated *MKI67* (**C-15** and **C-27**) represent chronically activated cells * TH17 cells (**C-12**, CCR6+ and *RORC*) * type 1 helper T cells (TH1; **C-17**, CXCR3+ and *IFNG* and *TBX21*) * heterogeneous continuum of intermediate TH1/TH17 states (**C-13**, **C-16** and **C-19**) with varying degrees of CXCR3, CCR6, CCR5 and CD161 surface protein expression and *RORC* and *TBX21* expression * CD161+ subset of type 2 helper T (TH2) cells (**C-14**), described as pathogenic, with higher expression of allergy-associated *HPGDS* and *IL17RB* * a subset of *FOXP3*+ Treg cells (**C-5**) expressing higher CCR6 surface protein and *CTLA4* and *RORC* than other Treg cells ![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-070418.png) ## 记忆T细胞的状态因人口特征和环境而异 发现： 年龄因素的影响最大；性别因素与T细胞状态也高度相关； type 2 helper T (TH2) 细胞状态在冬天（季节因素）搜集的样本中最为丰富 ![](https://media.springernature.com/full/springer-static/image/art%3A10.1038%2Fs41590-021-00933-1/MediaObjects/41590_2021_933_Fig3_HTML.png) ## 之后发现一个感兴趣的C-12状态 探索每个细胞状态和肺结核病情进展的关系，同时校正其他可能存在影响的因素（年龄、性别、季节、遗传以及其他技术因素），发现： **病人在肺结核发展时期，C-12显著降低了20%**，并且在年龄因素和男性因素下也会下降，而季节因素（冬季）会上升 ![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-073932.png) 又找到了**C-12 surface markers：CD4+CD26+CD161+CCR6+** ，这些marker已经被证明了以下功能： * IL-17-producing TH17 state * CD26 is a co-stimulatory molecule that promotes cytotoxicity * CD161 is associated with innate-like function * CCR6 is a homing marker that directs migration to inflamed sites ![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-075041.png) 那么C-12的下降是疾病的原因还是结果呢？ 利用外部数据进行验证，发现这种变化可能早于疾病发展 * predicted C-12 abundance was 9% lower in cases before progression compared to latent controls ![](https://jieandze1314-1255603621.cos.ap-guangzhou.myqcloud.com/blog/2021-05-25-082142.png)